The present invention relates to a DNA molecule encoding a variant human paraoxonase (EC 3.1.1.2), and to said variant paraoxonase protein. The present invention also relates to a method for detecting or predicting the risk of, or predisposition to, cancer, coronary and cerebrovascular diseases, type 2 diabetes, hypertension, dementia, arthrosis, cataract and sensitivity to organophosphorus compounds in a subject, as well as to a kit or assay for carrying out the said method. This invention also relates to targeting paraoxonase enhancing treatments and to transgenic animals comprising a human DNA molecule encoding said variant paraoxonase and to a method of mutation search.
The publications and other material used herein to illuminate the background of the invention are incorporated by reference.
Oxidative stress and free radicals have been implicated in the etiology of a number of diseases, including cancers, coronary heart disease, cerebrovascular disease, type 2 diabetes, hypertension, dementia and cataract. The human body has a number of endogenous free radicals scavenging systems which have genetic variability. The human serum paraoxonase (PON) is an enzyme carried in the high-density lipoprotein that contributes to the detoxification of organophosphorus compounds but also of toxic products of lipid peroxidation.1-9 The paraoxonase hydrolyzes the toxic metabolites of several organophosphorus (OP) insecticides, pesticides and nerve agents.
The PON1 gene is polymorphic in human populations and different individuals also express widely different levels and activities of the paraoxonase enzyme, which is the protein product coded by the gene.3,5-7 
Several polymorphisms are currently known in human PON1. The Gln191Arg poly-morphism was the first mutation of PON1 reported.3,6 The second one is the Met54Leu.3 Both these polymorphisms have been shown to affect serum PON activity.6,10,11 
Transgenic animals and with lowered paraoxonase activity can be used e.g. to test the effects of organophosphorus compounds, such as insecticides, pesticides and war agents, drugs that affect paraoxonase activity, other antioxidative compounds and drugs, and liver enzyme activity inducing agents.
A lot of methodological work has been done to locate disease-causing genes or candidate genes. However, there are no previous methodological studies concerning the methods of how to promote the search for mutations in a given or known candidate gene. To facilitate the finding of mutant DNA sequences, we developed a new method of phenotype-targeted gene sequencing.
One object of this invention is to provide a DNA sequence of a variant human PON1 gene and the amino acid sequence of the corresponding variant paraoxonase protein. Another object of the invention is to provide a method for screening a subject to assess if such subject is at risk of cancer, coronary or cerebrovascular disease, hypertension, type 2 diabetes, dementia, joint arthrosis or eye cataract, or at risk of being sensitive to organophosphate toxicity. The invention is also directed to a kit or an assay for said method, as well as to a probe for use in said method or kit. A further object of the invention is to provide a method for targeting a paraoxonase enhancing treatment for example for the above mentioned diseases and for organophospate poisoning, and/or for assessing the effectiveness of paraoxonase modifying treatments. A fourth object of the invention is to provide a transgenic animal with a gene encoding a variant paraoxonase. A fifth object of the invention is to provide a method for rapid search of gene mutations. These and further objects will be evident from the following description and claims.
According to one aspect, the invention concerns a DNA sequence comprising a nucleotide sequence encoding a variant paraoxonase protein with the Ile102Val mutation. The said mutation can, in the alternative, be named also Ile101 Val, if the start codon atg (Met) is not included in the count. In the following description and claims, reference is made to the Ile102Val mutation, but said reference means within the scope of the invention in the alternative the Ile101Val mutation in case the alternative way of counting is used. The invention also concerns a variant paraoxonase protein with the Ile102Val mutation.
According to further aspect, the invention concerns a method for screening a subject to determine if said subject is a carrier of a variant gene encoding a variant paraoxonase, by determining the allelic pattern for the codon 102 of the human PON1 gene, i.e. to determine if the said subject is a carrier of the Ile102Val mutation.
Specifically such a method comprises the steps of
a) providing a biological sample of the subject to be screened, and
b) providing an assay for detecting in the biological sample the presence of the Ile102Val or Val102Val genotype of the human PON1 gene.
The assay result can be used for assessing the subject""s risk to develop a low paraoxonase expression related disease such as cancer, coronary or cerebrovascular disease, type 2 diabetes, hypertension, dementia, arthrosis or cataract or sensitivity to organophosphorus compounds, and/or for assessing the effectiveness of paraoxonase-inducing therapy in a subject, whereby identification of a Ile102Val mutation being indicative of said risk being increased or effectiveness being modulated.
The present invention is thus directed to a method for detecting a risk of cancer, coronary or cerebrovascular disease, type 2 diabetes, hypertension, dementia, arthrosis or cataract in a subject, comprising isolating genomic DNA from said subject, determining the allelic pattern in the exon number 4 in the codon number 102 of the paraoxonase encoding PON1 gene in the genomic DNA, and identification of Ile102Val mutation indicating said risk being increased.
The present invention is also directed to a method for assessing the effectiveness of paraoxonase inducing therapy of a subject, comprising isolating genomic DNA from said subject, determining the allelic pattern in the exon number 4 in the codon number 102 of the paraoxonase encoding PON1 gene in the genomic DNA, and identification of Ile102Val mutation indicating said effectiveness being modulated, e.g. reduced.
The invention is also directed to a method for determining the presence or absence in a biological sample of a DNA sequence comprising a nucleotide sequence encoding a variant paraoxonase protein, the method comprising isolating genomic DNA from said subject, determining the allelic pattern in the exon number 4 in the codon number 102 of the paraoxonase encoding PON1 gene in the genomic DNA, and identification of Ile102Val mutation indicating the presence of said DNA sequence.
The techniques for carrying out such a method and presented here are intended to be non-limiting examples. One skilled in the art will readily appreciate that other methods for detection of the variant DNA sequence can be used, developed or modified.
One detection method is minisequencing which is based on a minisequencing reaction, in which an oligonucleotide that ends one nucleotide upstream the variant nucleotide, is enzymatically elongated by one nucleotide that is complementary to either the variant or the wild type nucleotide in the target sequence, and this added labelled nucleotide is detected. Such label can be, for example, radioactive or fluorescent label.
Another detection method is based on appearance or disappearance of an enzymatic cleavage site by the variant nucleotide. This kind of detection can be performed by first amplificating the target nucleotide sequence by a polymerase chain reaction with primers that flank the variant nucleotide, and then digesting the reaction product with a restriction endonuclease that recognises only the variant or only the wild-type sequence, producing DNA fragments of different length for each. These fragments may be recognised, for example, by gel electroforesis with DNA staining.
Yet another detection method is the oligonucleotide ligation assay, in which two allele specific oligonucleotide probes and one common oligonucleotide probe are used to distinguish between the variant and wild-type nucleotide. In this method, the target sequence is hybridised with the three oligonucleotide probes, and the probe pair that is complementary to the target sequence is joined enzymatically at the site of the variant nucleotide. The detection of the two alleles is based on differing labels, for example fluorescent labels of different colour, of the two allele specific oligonucleotide probes.
Furhermore, a detection method is the single stranded conformational analysis, in which the different alleles of a target sequence are identified on the basis of a difference in the electrophoretic mobility of the two alleles. In this method, the variant and wild-type target sequences that are in single stranded form, migrate with different speed through an electrophoresis matrix. Preferably, the target sequence is first amplified with a polymerase chain reaction, and the product is labelled for detection by radioactive or fluorescent label.
Yet furthermore, a detection method is sequencing, in which each nucleotide of the target sequence is identified. The variant allele is identified by the variant nucleotide.
Another detection method is allele specific hybridisation, in which an oligonucleotide probe is hybridised with the target sequence, and in which the probe is complementary only to the variant or wild-type allele. Preferably, two allele specific probes are used simultaneously to identify both alleles. Detection of a successful hybridisation and the determination of a genotype is based on detection of the probe-target duplex, on a basis of enzymatic colour reaction, or based on a label on the probe or on the target, for example a radioactive or a fluorescent label.
The present invention is also directed to a kit or assay for detecting a risk of cancer, coronary or cerebrovascular disease, type 2 diabetes, hypertension or dementia and sensitivity to organophosphorus compounds, and/or for assessing the need for or effectiveness of paraoxonase inducing therapy in a subject, comprising means for determining the allelic pattern in the exon number 4 in the codon 102 of the paraoxonase encoding PON1 gene in a genomic DNA sample. The assay may be a part of a DNA macroarray or microarray or a DNA chip or a DNA slide, which is intended for the detection of multiple gene mutations.
According to a further aspect, the present invention concerns a transgenic animal which carries a human DNA sequence comprising a nucleotide sequence encoding a variant human paraoxonase protein.
According to a further aspect, the present invention concerns the method of phenotype-targeted gene sequencing.